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P071 – Optimizing RNA Extraction and qPCR Analysis of Saliva Samples for Forensic Applications
Estimating the time since deposition (TSD) of bodily fluids can be crucial during a criminal investigation to verify witness statements or suspect’s alibi, establish relevance of the stain, and reconstruct the series of events. Context of evidence is important to an investigation so knowing the location of a fluid stain as well as the time it was deposited, provides context not previously available in criminal and sexual assault cases. Although numerous bodily fluids may be encountered at crime scenes, this study focuses on saliva due to its ease of collection and its prevalence at crime scenes or on a victim, especially in cases involving sexual assault. One potential method for estimating TSD involves assessing degradation patterns of different types of RNA. While previous research has been done and degradation rates have shown potential for valuable insight in estimating the age of a stain, there are currently no accepted or reliable methods. The purpose of this research was to use deposited saliva stains to optimize the process of RNA extraction, quantification, reverse transcription, and RT-PCR, in order to develop a reliable TSD method in the future.
Due to the nature of saliva and the large presence of a microbiome, it is important to ensure human specificity which can be done during reverse transcription. Evaluation of targets that are universally and consistently expressed across different tissues and physiological states or the organism are important for optimization studies, so primers were designed for four different housekeeping genes (HKGs) and used during RT-PCR to create a baseline for comparison and be able to identify issues related to sample preparation or experimental design. Quantitation results indicated success of the extraction method for experimental samples, amplification results from the cDNA standard confirmed the qPCR setup and results of the reverse-transcribed RNA control confirmed proper function of the reverse transcription kit, and both standards for use as controls. Although the experimental samples unfortunately failed to produce amplification with the selected primers, consistent performance of both positive and negative controls was achieved. However, the B2M primer showed promising results for future analyses with some amplification present within two experimental samples and no amplification in the negative controls.
Author
- Colleen Pastore (Syracuse University- Forensic & National Security Sciences Institute, United States of America)
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